We studied three site-directed mutants of annexin XII (E105K, E105K/K68A, E105K/K68A/E76A), aimed at investigating the roles of amino acid residues participating in the intermolecular interaction which plays an important role in the Ca2+-induced hexamer formation. We observed significantly smaller radii of gyration with E105K in the presence of saturated amounts of Ca2+ as compared to the wild type protein in an identical solution condition. The hexamer formation is significantly diminished for the double and triple mutants as expected from the crystal structure. Thus we conclude that these amino acid residues are crucial in the Ca2+-induced oligomer formation.